The Flavivirus genus of the Flaviviridae family includes approximately 70 viruses, mostly arboviruses, many of which, such as yellow fever (YF), dengue (DEN), Japanese encephalitis (JE), and tick-borne encephalitis (TBE) viruses, are major human pathogens (rev. in Burke and Monath, Fields Virology, 4th Ed.: 1043-1126, 2001). For example, Japanese encephalitis is the leading cause of viral encephalitis in Asia, where 30,000 to 50,000 new cases are reported each year. As another example, since the first cases were diagnosed in the New York area in 1999, West Nile virus has continued to spread rapidly across North America. The risks of this virus migrating into South America, as well as an epidemic in underdeveloped countries, are extremely high. Effective methods for preventing infection by these viruses are needed, with vaccination being the most cost effective measure.
The Flavivirus particle contains a nucleocapsid composed of viral RNA and capsid protein C. The nucleocapsid is surrounded by an envelope containing the envelope glycoprotein E (50-60 kDa) and a small membrane protein M (7-8 kDa). Translation of the genomic RNA results in a polyprotein precursor that is cleaved by cellular and viral proteases into viral proteins, in the order: C, prM/M, E, NS 1, NS2A, NS2B, NS3, NS4A, 2K, NS4B, and NS5, where C through E are the structural components of the virion and NS 1 through NS5 are nonstructural proteins required for replication (Lindenbach and Rice, Fields Virology, 4th Ed.:991-1041, 2001). The prM protein (˜25 kDa) is the intracellular precursor for M. Immature virions containing prM are produced by budding into the lumen of the endoplasmic reticulum (ER) and are transported to the cell surface through the exocytosis pathway. Cleavage of prM occurs shortly prior to particle release in post-Golgi vesicles. Mature extracellular virus contains predominantly M protein, although a small fraction of uncleaved prM can also be present.
The E protein is the main functional and antigenic surface component of the virion. The molecular structure of the ectodomain of E, which forms a homodimer on the surface of mature viral particles at neutral pH, has been resolved by cryoelectron microscopy (Rey et al., Nature 375:291-298, 1995) and fitted into the electron density map of viral particles (Kuhn et al., Cell 108:717-725, 2002). During infection, the E protein functions as a class II fusion protein (Modis et al., Nature 427:313-319, 2004). Following virus binding to a cellular receptor and internalization, the acidic pH in the resulting endosomes triggers dissociation of the dimers such that the previously hidden hydrophobic fusion loop of each monomer is exposed outwardly. Concurrently, the loops insert into the cell (endosome) membrane and monomers rearrange into elongated trimers. Further refolding of the trimers brings the cell and viral membranes into close proximity and forces them to fuse, releasing the contents of the viral particle into the cytoplasm. Previous studies showed that some substitutions in the E protein of DEN and JE, which are selected during serial passages in mouse brain and in cultured monkey kidney and mosquito cells, have been localized in particular regions of the 3D structure of the protein, and were reported to be associated with changes in the fusion function of the viruses. The studies showed that the fusion pH threshold for some attenuated vaccines decreased by 0.6 to 1 pH unit by comparison with the corresponding parental virus isolate. Some changes in six residues in the DEN3 protein E (residues 54, 191, 202, 266, 268, and 277) map to the region in domain II. This region is proposed as a focus for the low-pH mediated confiormational change required for the surface exposure of the conserved hydrophobic cd fusion loop (Lee et al., Virology 232:281-290, 1997).
There is no evidence that the small (mature) M protein plays a role in the events leading to virus internalization from the endosome or has any other appreciable function, while its intracellular precursor, prM, is known to be important for morphogenesis and transport of progeny viral particles. The prM protein also facilitates proper folding of E (Lorenz et al., J. Virol. 76:5480-5491, 2002) and functions to protect the E protein dimer from premature conformational rearrangement during passage of new particles towards the cell surface through acidic secretory compartments (Guirakhoo et al., J. Gen. Virol. 72:1323-1329, 1991; Guirakhoo et al., Virology 191:921-931, 1992).
ChimeriVax™ technology has been used to create live, attenuated vaccine candidates against medically important Flaviviruses. It employs the YF 17D vaccine virus as a vector in which the prM-E genes are replaced with the prM-E genes from a heterologous Flavivirus, such as JE, dengue, West Nile, or St. Louis encephalitis viruses (Monath et al., Vaccine 17:1869-1882, 1999; Monath et al., Curr. Drug Targets—Inf. Disorders 1:37-50, 2001; Monath et al., Vaccine 20:1004-1018, 2002; Guirakhoo et al., Virology 257:363-372, 1999; Guirakhoo et al., J. Virol. 75:7290-7304, 2001; Guirakhoo et al., Virology 298:146-159, 2002; Pugachev et al., Int. J. Parasitol. 33:567-582, 2003; Guirakhoo et al., J. Virol. 78:4761-4775, 2004). Previously, the ChimeriVax™-JE vaccine virus, containing the prM-E genes from the SA14-14-2 virus (live attenuated JE vaccine used in China), was propagated to high titers in Vero cells cultured in media supplemented with fetal bovine serum (FBS) (Monath et al., Biologicals 33:131-144, 2005). It was successfully tested in preclinical and Phase I and II clinical trials (Monath et al., Vaccine 20:1004-1018, 2002; Monath et al., J. Infect. Dis. 188:1213-1230, 2003). Similarly, successful Phase I clinical trials have been conducted with a ChimeriVax™-WN vaccine candidate, which contains the prM-E sequence from a West Nile virus (NY99 strain), with three specific amino acid changes incorporated into the E protein to increase attenuation (Arroyo et al., J. Virol. 78:12497-12507, 2004).